Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

We study the mechanobiology underlying tendon impingement and the process by which this unique mechanical demand drives localized fiber cartilage formation in health and disease. Here we seek to characterize matrix for modeling relative to spatially heterogeneous patterns of multi axial mechanical strain generated by impingement and to identify molecular mechanisms mediating this response. In vitro models for studying impingement mechanobiology have applied simple compression to isolated tendon cells or artificial uniaxial compression to partial and whole tendon explants.

Established animal models of tendon impingement manipulating external source of tendon impingement in vivo most often surgically and explored biology following resumed physical activity. In vitro models present significant limitations as isolated cells lack their three dimensional extracellular environment crucial to McKenna response. While excised explant models circumvent this limitation, both fail to recreate multiaxial strain patterns generated by impingement in vivo.

Conversely, animal models offer limited ability to measure or control internal tissue strains. Our murine hind limb explant model for studying impingement mechanobiology maintain cells within their extracellular environment and preserves local anatomy of the impinged Achilles tendon insertion in situ, allowing controlled prescription of impingement through passively applied joint motion to recreate multiaxial patterns of tissue strain that are measurable and well-characterized. To begin, transfer 70 milliliters of prewarmed Dulbecco's modified Eagle medium into a Petri dish in a sterile biosafety cabinet and add dexamethasone at a concentration of 100 nanomolar.

Assemble the surgical tools required for the dissection, which includes smooth, straight fine-tip forceps, straight fine-tip forceps with serrated teeth, and straight, sharp, fine scissors. To dissect hind limb explants locate the hip joint of a euthanized mouse positioned supine on the benchtop lined with absorbent underpads. Use fine scissors to create a small incision on the skin covering the proximal and anterior aspects of the upper leg.

Then, use fingers to pull the incision apart to expand and pinch the exposed upper leg. Carefully pull the skin distally to deglove the hind limb to the level of the ankle. Insert one scissor blade gently under the skin along the dorsal aspect of the foot, then make an incision that extends towards the toes and continue pulling the skin distally until it is completely removed.

Position the mouse to have a clear view of the Achilles tendon insertion onto the posterior aspect of the calcaneus near the ankle. To remove the plantaris tendon, insert one tip of the smooth, fine-tip forceps between the two tendons, pass the tip medially under the plantaris tendon, then pull it proximally to tear through the plantaris muscle. Then using fine-tip serrated forceps, grasp the detached proximal end of the plantaris tendon and pull it distally to completely remove it.

At the hip joint, employ fine scissors to precisely sever through the pelvis, isolating the hind limb. Use the scissors to gently remove the remaining pelvis, revealing the femoral head. In the biosafety cabinet, transfer the hind limb explant into the dish containing cell culture media and dexamethasone.

Relocate the dish to the incubator and subject it to pre-treatment for 48 hours. To begin, preheat an adequate amount of sterile culture media while the 48-hour dexamethasone pretreatment of murine hind limb explant is about to complete. Once the pretreatment ends, move the pretreated explants to the biosafety cabinet.

for the unloaded group, aspirate the pretreatment media and transfer the explants to fresh Petri dishes. Add 70 milliliters of culture media to each dish before returning them to the incubator. To prepare the explant platforms for the baseline tension and static impingement group cut pieces of sandpaper to match the size of the grip platens.

For the static impingement group, cut pieces of braided line and pre-tie a loose overhand knot halfway along the length of the line. Tear pieces of aluminum foil to cover each acrylic bath and spray them with 70%ethanol. Transfer the prepared components to the biosafety cabinet.

Arrange the components such as acrylic bath, base, volume reduction inserts, clip, and grips for platform assembly. Place platform components inside secondary containers of sufficient volume to contain any potential culture media leaks. Submerge the containers in a solution of at least 10%bleach for a minimum of one hour.

Rinse the bleach solutions with tap water. After transferring the rinsed components to the biosafety cabinet, use M3 screws and compressions springs to affix the platens to the grips. Securely attach the grips to the base employing an M5 screw and insert the M6 screws until they contact the platens.

Enhance the platens functionality by affixing sandpaper using double-sided tape. Close the grips to facilitate the adhesion of the sandpaper to the platens. To load a platform, fully open the grips and use forceps to carefully position the upper leg and knee between the platens.

Close the grips gently to hold the limb in place. Use forceps to grasp the exposed femoral head or foot and adjust the knee flexion angle while gradually closing the forceps to secure it in place. For the baseline tension group, extend the knee joint while closing the grips.

In the case of the static impingement group, flex the knee joint while closing the grips. For the static impingement group, place the overhand knot of the string around the distal paw and tighten it securely. Route the string through a slot in the base underneath the explant and through the clip hole.

Position the base into the acrylic bath and fasten the clip to the top edge of the bath. To dorsiflex the foot pull the string until it reaches at least 110 degrees relative to the tibia. Use a permanent marker to mark the string as it exits the clip.

Capture a photograph of the explant in this dorsiflexed position for future quantification of the dorsiflexion angle. Remove the base from the setup and attach the volume reduction insert by sliding it along a track on the base. Place the base back into the acrylic bath and reposition the clip on the top edge.

Use the marked string as a guide. Pull it to restore the foot to its original dorsiflexion angle and secure the string to the exterior of the bath using tape to maintain a static dorsiflexed position. For the baseline tension group, once the explant is positioned between the grips, take a photograph to quantify the dorsiflexion angle, then slide the volume reduction insert onto the base and place it into the acrylic bath.

Add 125 milliliters of prewarmed culture media to each platform to submerge the explants. After covering the top of the bath with aluminum foil, place the entire setup into a secondary container. Relocate the secondary container to the incubator and culture for seven days.